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Abstract:

The present invention includes compositions and methods for making and
using anti DC-ASGPR antibodies that can, e.g., activate DCs and other
cells.

Claims:

1. A method for increasing the effectiveness of antigen presentation
comprising the step of isolating and purifying a DC-ASGPR-specific
antibody or fragment thereof to which an antigen is attached that forms
an antibody-antigen complex, wherein the antigen is processed and
presented by a dendritic cell that has been contacted with the
antibody-antigen complex.

3. The method of claim 1, wherein DC-ASGPR-specific antibody or fragment
thereof is bound to one half of a Coherin/Dockerin pair.

4. The method of claim 1, wherein DC-ASGPR-specific antibody or fragment
thereof is bound to one half of a Coherin/Dockerin pair and an antigen is
bound to the complementary half of the Coherin/Dockerin pair to form a
complex.

5. The method of claim 1, wherein the antigen is selected from a peptide,
protein, lipid, carbohydrate, nucleic acid, and combinations thereof.

8. A method for increasing the effectiveness of antigen presentation by
dendritic cells comprising binding a DC-ASGPR-specific antibody or
fragment thereof to which an antigen is attached that forms an
antibody-antigen complex, wherein the antigen is processed and presented
by a dendritic cell that has been contacted with the antibody-antigen
complex.

9. The method of claim 8, wherein increased effectiveness of the dendritic
cells is determined used allogeneic CD8.sup.+ T cells.

10. The use of antibodies or other specific binding molecules directed to
DC-ASGPR for delivering antigens to antigen-presenting cells for the
purpose of eliciting protective or therapeutic immune responses.

11. The use of antigen-targeting reagents specific to DC-ASGPR for
vaccination via the skin.

12. The use of antigen-targeting reagents specific to DC-ASGPR in
association with co-administered or linked adjuvant for vaccination.

13. The use for antigen-targeting (vaccination) purposes of specific
antigens which can be expressed as recombinant antigen-antibody fusion
proteins.

14. An antigen specific anti-DC-ASGPR immunoglobulin or fragment thereof
that is secreted from mammalian cells and an antigen bound to the
immunoglobulin.

15. The immunoglobulin of claim 14, wherein the antigen is a fusion
protein with the immunoglobulin.

17. The immunoglobulin of claim 14, wherein the immunoglobulin is bound to
one half of a cohesin/dockerin domain.

18. The immunoglobulin of claim 14, further comprising a complementary
half of the cohesin-dockerin binding pair bound to an antigen that forms
a complex with the modular rAb carrier.

19. The immunoglobulin of claim 14, further comprising a complementary
half of the cohesin-dockerin binding pair that is a fusion protein with
an antigen.

20. The immunoglobulin of claim 14, wherein the antigen specific domain
comprises a full length antibody, an antibody variable region domain, an
Fab fragment, a Fab' fragment, an F(ab)2 fragment, and Fv fragment,
and Fabc fragment and/or a Fab fragment with portions of the Fc domain.

25. A method for increasing the effectiveness of dendritic cells
comprising:isolating patient dentritic cellsexposing the dendritic cells
to activating amounts of anti-DC-ASGPR antibodies or fragments thereof
and antigen to form antigen-loaded, activated dendritic cells;
andreintroducing the antigen-loaded, activated dendritic cells into the
patient.

26. Use of agents that engage DC-ASGPR, alone or with co-activating
agents, to activate antigen-presenting cells for therapeutic or
protective applications.

27. The method of claim 25, wherein the DC-ASGPR binding and/or activating
agents are linked to antigens, alone or with co-activating agents, for
protective or therapeutic vaccination.

28. The method of claim 25, wherein the specific antibody V-region
sequences is capable of binding to and activating DC-ASGPR.

29. Use of anti-DC-ASGPR agents linked to toxic agents for therapeutic
purposes in the context of diseases known or suspected to result from
inappropriate activation of immune cells via DC-ASGPR.

30. A vaccine comprising a DC-ASGPR-specific antibody or fragment thereof
to which an antigen is attached that forms an antibody-antigen complex,
wherein the antigen is processed and presented by a dendritic cell that
has been contacted with the antibody-antigen complex.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims priority to U.S. Provisional Application
Ser. No. 60/888,036, filed Feb. 2, 2007, the content of which is
incorporated by reference herein in its entirety.

TECHNICAL FIELD OF THE INVENTION

[0003]The present invention relates in general to the field of agents that
engage antigen-presenting cells through dendritic cell asialoglycoprotein
receptor (DC-ASGPR).

BACKGROUND OF THE INVENTION

[0004]Without limiting the scope of the invention, its background is
described in connection with antigen presentation.

[0005]Dendritic Cells play a pivotal role in controlling the interface of
innate and acquired immunity by providing soluble and intercellular
signals, followed by recognition of pathogens. These functions of DCs are
largely dependent on the expression of specialized surface receptors,
`pattern recognition receptors` (PRRs), represented, most notably, by
toll-like receptors (TLRs) and C-type lectins or lectin-like receptors
(LLRs) (1-3).

[0006]In the current paradigm, a major role of TLRs is to alert DCs to
produce interleukin 12 (IL-12) and other inflammatory cytokines for
initiating immune responses. C-type LLRs operate as constituents of the
powerful antigen capture and uptake mechanism of macrophages and DCs (1).
Compared to TLRs, however, LLRs might have broader ranges of biological
functions that include cell migrations (4), intercellular interactions
(5). These multiple functions of LLRs might be due to the facts that
LLRs, unlike TLRs, can recognize both self and nonself. However, the
complexity of LLRs, including the redundancy of a number of LLRs
expressed in immune cells, has been one of the major obstacles to
understand the detailed functions of individual LLRs. In addition,
natural ligands for most of these receptors remain unidentified.
Nonetheless, evidence from recent studies suggests that LLRs, in
collaboration with TLRs, may contribute to the activation of immune cells
during microbial infections (6-14).

[0007]Valladeau et al. (The Journal of Immunology, 2001, 167: 5767-5774)
described a novel LLR receptor on immature human Dendritic Cells related
to hepatic Asialoglycoprotein Receptor and demonstrated that it
efficiently mediated endocytosis. DC-ASGPR mRNA was observed
predominantly in immune tissues--in DC and granulocytes, but not in T, B,
or NK cells, or monocytes. DC-ASGPR species were restricted to the CD
14-derived DC obtained from CD34-derived progenitors, while absent from
the CD1a-derived subset. Both monocyte-derived DC and tonsillar
interstitial-type DC expressed DC-ASGPR protein, while Langerhans-type
cells did not. Furthermore, DC-ASGPR was a feature of immaturity, as
expression was lost upon CD40 activation. In agreement with the presence
of tyrosine-based and dileucine motifs in the intracytoplasmic domain,
mAb against DC-ASGPR was rapidly internalized by DC at 37° C.
Finally, intracellular DC-ASGPR was localized to early endosomes,
suggesting that the receptor recycles to the cell surface following
internalization of ligand. These findings identified DC-ASGPR/human
macrophage lectin as a feature of immature DC, and as another lectin
important for the specialized Ag-capture function of DC.

SUMMARY OF THE INVENTION

[0008]While DC-ASGPR is known to be capable of directing the
internalization of surrogate antigen into human DC, the invention uses
novel biological activities of DC-ASGPR to effect particularly desirable
changes in the immune system, some in the context of antigen uptake
(e.g., vaccination), others through the unique action of DC-ASGPR
effectors (alone or in concert with other immune regulatory molecules)
capable of eliciting signaling through this receptor on DC, B cells, and
monocytes. The invention disclosure reveals means of developing unique
agents capable of activating cells bearing DC-ASGPR, as well as the
effect of the resulting changes in cells receiving these signals regards
action on other cells in the immune system. These effects (either alone,
or in concert with other signals (i.e., co-stimulation)) are highly
predictive of therapeutic outcomes for certain disease states or for
augmenting protective outcomes in the context of vaccination.

[0009]The present invention includes compositions and methods for
increasing the effectiveness of antigen presentation by a
DC-ASGPR-expressing antigen presenting cell by isolating and purifying a
DC-ASGPR-specific antibody or fragment thereof to which a targeted agent
is attached that forms an antibody-antigen complex, wherein the agent is
processed and presented by, e.g., a dendritic cell, that has been
contacted with the antibody-agent complex. In one embodiment, the antigen
presenting cell is a dendritic cell and the DC-ASGPR-specific antibody or
fragment thereof is bound to one half of a Coherin/Dockerin pair. The
DC-ASGPR-specific antibody or fragment thereof may also be bound to one
half of a Coherin/Dockerin pair and an antigen is bound to the
complementary half of the Coherin/Dockerin pair to form a complex.
Non-limiting examples agents include one or more peptides, proteins,
lipids, carbohydrates, nucleic acids and combinations thereof.

[0011]The present invention also includes compositions and methods for
increasing the effectiveness of antigen presentation by dendritic cells
comprising binding a DC-ASGPR-specific antibody or fragment thereof to
which an antigen is attached that forms an antibody-antigen complex,
wherein the antigen is processed and presented by a dendritic cell that
has been contacted with the antibody-antigen complex. Another embodiment
is the use of antibodies or other specific binding molecules directed to
DC-ASGPR for delivering antigens to antigen-presenting cells for the
purpose of eliciting protective or therapeutic immune responses. The use
of antigen-targeting reagents specific to DC-ASGPR for vaccination via
the skin; antigen-targeting reagents specific to DC-ASGPR in association
with co-administered or linked adjuvant for vaccination or use for
antigen-targeting (vaccination) purposes of specific antigens which can
be expressed as recombinant antigen-antibody fusion proteins.

[0012]Another embodiment includes a method for increasing the
effectiveness of dendritic cells by isolating patient dendritic cells;
exposing the dendritic cells to activating amounts of anti-DC-ASGPR
antibodies or fragments thereof and antigen to form antigen-loaded,
activated dendritic cells; and reintroducing the antigen-loaded,
activated dendritic cells into the patient. The antigen may be a
bacterial, viral, fungal, protozoan or cancer protein. The present
invention also includes an anti-DC-ASGPR immunoglobulin or portion
thereof that is secreted from mammalian cells and an antigen bound to the
immunoglobulin. The immunoglobulin is bound to one half of a
cohesin/dockerin domain, or it may also include a complementary half of
the cohesin-dockerin binding pair bound to an antigen that forms a
complex with the modular rAb carrier, or a complementary half of the
cohesin-dockerin binding pair that is a fusion protein with an antigen.
The antigen specific domain may be a full length antibody, an antibody
variable region domain, an Fab fragment, a Fab' fragment, an F(ab)2
fragment, and Fv fragment, and Fabc fragment and/or a Fab fragment with
portions of the Fc domain. The anti-DC-ASGPR immunoglobulin may also be
bound to a toxin selected from wherein the toxin is selected from the
group consisting of a radioactive isotope, metal, enzyme, botulin,
tetanus, ricin, cholera, diphtheria, aflatoxins, perfringens toxin,
mycotoxins, shigatoxin, staphylococcal enterotoxin B, T2, seguitoxin,
saxitoxin, abrin, cyanoginosin, alphatoxin, tetrodotoxin, aconotoxin,
snake venom and spider venom. The antigen may be a fusion protein with
the immunoglobulin or bound chemically covalently or not.

[0013]The present invention also includes compositions and methods for
increasing the effectiveness of dendritic cells by isolating patient
dendritic cells, exposing the dendritic cells to activating amounts of
anti-DC-ASGPR antibodies or fragments thereof and antigen to form
antigen-loaded, activated dendritic cells; and reintroducing the
antigen-loaded, activated dendritic cells into the patient. The agents
may be used to engage DC-ASGPR, alone or with co-activating agents, to
activate antigen-presenting cells for therapeutic or protective
applications, to bind DC-ASGPR and/or activating agents linked to
antigens, alone or with co-activating agents, for protective or
therapeutic vaccination. Another use of is the development of specific
antibody V-region sequences capable of binding to and activating
DC-ASGPR, for use as anti-DC-ASGPR agents linked to toxic agents for
therapeutic purposes in the context of diseases known or suspected to
result from inappropriate activation of immune cells via DC-ASGPR and as
a vaccine with a DC-ASGPR-specific antibody or fragment thereof to which
an antigen is attached that forms an antibody-antigen complex, wherein
the antigen is processed and presented by a dendritic cell that has been
contacted with the antibody-antigen complex.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]For a more complete understanding of the features and advantages of
the present invention, reference is now made to the detailed description
of the invention along with the accompanying figures and in which:

[0015]FIGS. 1A to 1E demonstrate signaling through lectin-like receptor
DC-ASGPR activates DCs, resulting in increased levels of costimulatory
molecules as well as cytokines and chemokines. FIG. 1A shows three day
and six day GM/IL-4 DCs were stained with FITC-labeled goat anti-mouse
IgG followed by mouse monoclonal anti-human DC-ASGPR, antibody. FIG. 1B
shows six day GM/IL-4 DCs were cultured in plates coated with the
anti-DC-ASGPR or control mAbs (1-2 μg/ml) for 16-18 h. Cells were
stained with anti-CD86 and HLA-DR antibodies labeled with fluorescent
dyes. Open and filled bars in the histograms represent cells activated
with isotype control mAbs and anti-lectin mAbs, respectively. FIG. 1C
shows six day GM/IL-4 DCs were cultured in plates coated with the mAbs
for 12 h, and subjected to RNA isolation and Affymetrix Gene Chip
analysis, as described in Methods. Fold increases of gene expression by
anti-lectin mAbs were compared with the gene expression levels in DCs
stimulated with control mAbs. FIG. 1D shows the cytokines and chemokines
in the culture supernatants from the experiment shown in FIG. 1B were
measured by Luminex. FIG. 1E shows six day GM/IL-4 DCs were cultured in
plates coated with mAbs in the presence or absence of 50 ng/ml soluble
CD40L, for 16-18 h, and then stained with anti-CD83 antibodies. Cytokines
and chemokines in the culture supernatants from the experiment shown in
FIG. 1E were measured by Luminex. Results shown are representative of
three independent experiments using cells from different normal donors.

[0016]FIGS. 2A to 2D shows that DC-ASGPR expressed on DCs, contributes to
enhanced humoral immune responses. Six day GM/IL-4 DCs,
5×103/well, were incubated in 96 well plates coated with
anti-DC-ASGPR or control mAb for 16-18 h, and then 1×105
autologous CD19+ B cells stained with CFSE were co-cultured in the
presence of 20 units/ml IL-2 and 50 nM CpG. FIG. 2A is a FACS of day six
cells stained with fluorescently labeled antibodies. CD3+and
7-AAD+ cells were gated out. CD38+ and CFSE.sup.- cells were
purified by FACS sorter and Giemsa staining was performed. FIG. 2B are
culture supernatants on day thirteen were analyzed for total IgM, IgG,
and IgM by sandwich ELISA. FIG. 1C shows DCs pulsed with 5 multiplicity
of infection (moi) of heat-inactivated influenza virus (PR8), and
cultured with B cells. Culture supernatant was analyzed for
influenza-specific immunoglobulins (Igs) on day thirteen. FIG. 1D shows
DC cultured with anti-DC-ASGPR or control mAb were stained for cell
surface APRIL expression and the supernatants assayed for soluble APRIL.

[0017]FIGS. 3A to 3D shows the cell surface expression of DC-ASGPR on B
cells contribute to B cell activation and immunoglobulin production. FIG.
3A are PBMCs from buffy coats were stained with anti-CD19, anti-CD3, and
anti-DC-ASGPR or control mAb. CD19+ and CD3+ cells were gated
and the expression levels of the molecules on CD19+ B cells were
measured by flow cytometry. FIG. 3B are CD19+ B cells from buffy
coats were cultured in plates coated with the mAbs for 12 h, and
subjected to RNA isolation and Affymetrix Gene Chip analysis as described
in Methods. Fold increases of gene expression by anti-DC-ASGPR mAb were
compared to the gene expression levels in CD19+ B cells stimulated
with control mAb. FIG. 3c shows CD19+ B cells were cultured in
plates coated with the mAbs for 16-18 h, and then culture supernatants
were analyzed for cytokines and chemokines by Luminex. FIG. 3D shows
1×105 CD19+ B cells were cultured in plates coated with
the mAbs for thirteen days. Total Ig levels were measured by ELISA. Data
are representative of two repeat experiments using cells from three
different normal donors.

[0018]FIGS. 4A to 4D shows that the proliferation of purified allogeneic T
cells was significantly enhanced by DCs stimulated with mAb specific for
DC-ASGPR.

[0019]FIG. 5 shows that certain anti-DC-ASGPR mAbs can activate DC.
GM-CSF/IL-4. DC were incubated for 24 hrs with one of a panel of 12 pure
anti-ASGPR mAbs. Cells were then tested for expression of cell surface
CD86 (a DC activation marker) and supernatants were assayed for secreted
cytokines. Three mAbs (36, 38, 43) from the anti-ASGPR mAb panel
activated DC.

[0020]FIG. 6 shows that different antigens can be expressed in the context
of a DC-ASGPR rAb. Such an anti-DC-ASGPR rAb.Doc protein can be simply
mixed with any Cohesin.fusion protein to assemble a stable non-covalent
[rAb.Doc:Coh.fusion] complex that functions just as a rAb.fusion protein.

[0021]FIG. 7--GM-CSF/IFNa DCs (5,000/well) were loaded with 10 or 1 nM
anti-DC-ASGPR.Doc:Coh.Flu M1, or hIgG4.Doc:Coh.Flu M1 complexes. After 6
h, autologous CD8+ T cells (200,000/well) were added into the cultures.
At day 8, the CD8+ T cells were analyzed for expansion of cells bearing
TCR specific for a HLA-A201 immuno-dominant peptide. The inner boxes
indicate the percentage of tetramer-specific CD8+ T cells.

[0022]FIG. 8 demonstrated the cross reactivity of the different antibodies
with monkey ASGPR.

DETAILED DESCRIPTION OF THE INVENTION

[0023]While the making and using of various embodiments of the present
invention are discussed in detail below, it should be appreciated that
the present invention provides many applicable inventive concepts that
can be embodied in a wide variety of specific contexts. The specific
embodiments discussed herein are merely illustrative of specific ways to
make and use the invention and do not delimit the scope of the invention.

[0024]To facilitate the understanding of this invention, a number of terms
are defined below. Terms defined herein have meanings as commonly
understood by a person of ordinary skill in the areas relevant to the
present invention. Terms such as "a", "an" and "the" are not intended to
refer to only a singular entity, but include the general class of which a
specific example may be used for illustration. The terminology herein is
used to describe specific embodiments of the invention, but their usage
does not delimit the invention, except as outlined in the claims.

[0025]Dendritic cells (DCs) are antigen-presenting cells that play a key
role in regulating antigen-specific immunity (Mellman and Steinman 2001),
(Banchereau, Briere et al. 2000), (Cella, Sallusto et al. 1997). DCs
capture antigens, process them into peptides, and present these to T
cells. Therefore delivering antigens directly to DC is a focus area for
improving vaccines. One such example is the development of DC-based
vaccines using ex-vivo antigen-loading of autologous DCs that are then
re-administrated to patients (Banchereau, Schuler-Thurner et al. 2001),
(Steinman and Dhodapkar 2001). Another strategy to improve vaccine
efficacy is specific targeting to DC of antigen conjugated to antibodies
against internalizing DC-specific receptors. The potential of targeting
DC for vaccination is highlighted by key mouse studies. In vivo,
targeting with an anti-LOX-1 mAb coupled to ovalbumin (OVA) induced a
protective CD8+ T cell response, via exogenous antigen cross-presentation
toward the MHC class I pathway (Delneste, Magistrelli et al. 2002). Also,
OVA conjugated to anti-DEC205 mAb in combination with a CD40L maturation
stimulus enhanced the MHC class I-restricted presentation by DCs in vivo
and led to the durable formation of effector memory CD8+ T cells
(Bonifaz, Bonnyay et al. 2004). Both these studies showed dramatic
dose-sparing (i.e., strong immune-responses at very low antigen doses)
and suggested broader responses than normally seen with other types of
OVA immunization. Recent work with targeting of HIV gag antigen to DC via
DEC205 has extended these concepts to a clinically relevant antigen and
confirmed the tenants of targeting antigen to DC--dramatic dose-sparing,
protective responses from a single vaccination, and expansion of
antigen-specific T cells in both the CD8 and CD4 compartments
(Trumpfheller, Finke et al. 2006).

[0026]The present invention provides for the complexing of multiple
antigens or proteins (engineered, expressed, and purified independently
from the primary mAb) in a controlled, multivariable fashion, to one
single primary recombinant mAb. Presently, there are methods for
engineering site-specific biotinylation sites that provide for the
addition of different proteins (each engineered separately linked to
streptavidin) to the one primary mAb. However, the present invention
provides for addition to the primary mAb of multiple combinations, in
fixed equimolar ratios and locations, of separately engineered proteins.

[0027]As used herein, the term "modular rAb carrier" is used to describe a
recombinant antibody system that has been engineered to provide the
controlled modular addition of diverse antigens, activating proteins, or
other antibodies to a single recombinant monoclonal antibody (mAb). The
rAb may be a monoclonal antibody made using standard hybridoma
techniques, recombinant antibody display, humanized monoclonal antibodies
and the like. The modular rAb carrier can be used to, e.g., target (via
one primary recombinant antibody against an internalizing receptor, e.g.,
a human dendritic cell receptor) multiple antigens and/or antigens and an
activating cytokine to dendritic cells (DC). The modular rAb carrier may
also be used to join two different recombinant mAbs end-to-end in a
controlled and defined manner.

[0028]The antigen binding portion of the "modular rAb carrier" may be one
or more variable domains, one or more variable and the first constant
domain, an Fab fragment, a Fab' fragment, an F(ab)2 fragment, and Fv
fragment, and Fabc fragment and/or a Fab fragment with portions of the Fc
domain to which the cognate modular binding portions are added to the
amino acid sequence and/or bound. The antibody for use in the modular rAb
carrier can be of any isotype or class, subclass or from any source
(animal and/or recombinant).

[0029]In one non-limiting example, the modular rAb carrier is engineered
to have one or more modular cohesin-dockerin protein domains for making
specific and defined protein complexes in the context of engineered
recombinant mAbs. The mAb is a portion of a fusion protein that includes
one or more modular cohesin-dockerin protein domains carboxy from the
antigen binding domains of the mAb. The cohesin-dockerin protein domains
may even be attached post-translationally, e.g., by using chemical
cross-linkers and/or disulfide bonding.

[0030]The term "antigen" as used herein refers to a molecule that can
initiate a humoral and/or cellular immune response in a recipient of the
antigen. Antigen may be used in two different contexts with the present
invention: as a target for the antibody or other antigen recognition
domain of the rAb or as the molecule that is carried to and/or into a
cell or target by the rAb as part of a dockerin/cohesin-molecule
complement to the modular rAb carrier. The antigen is usually an agent
that causes a disease for which a vaccination would be advantageous
treatment. When the antigen is presented on MHC, the peptide is often
about 8 to about 25 amino acids. Antigens include any type of biologic
molecule, including, for example, simple intermediary metabolites,
sugars, lipids and hormones as well as macromolecules such as complex
carbohydrates, phospholipids, nucleic acids and proteins. Common
categories of antigens include, but are not limited to, viral antigens,
bacterial antigens, fungal antigens, protozoal and other parasitic
antigens, tumor antigens, antigens involved in autoimmune disease,
allergy and graft rejection, and other miscellaneous antigens.

[0032]Examples of anti-tumor agents for delivery using the present
invention include, without limitation, doxorubicin, Daunorubicin, taxol,
methotrexate, and the like. Examples of antipyretics and analgesics
include aspirin, Motrin®, Ibuprofen®, naprosyn, acetaminophen,
and the like.

[0033]Examples of anti-inflammatory agents for delivery using the present
invention include, without limitation, include NSAIDS, aspirin, steroids,
dexamethasone, hydrocortisone, prednisolone, Diclofenac Na, and the like.

[0034]Examples of therapeutic agents for treating osteoporosis and other
factors acting on bone and skeleton include for delivery using the
present invention include, without limitation, calcium, alendronate, bone
GLa peptide, parathyroid hormone and its active fragments, histone
H4-related bone formation and proliferation peptide and mutations,
derivatives and analogs thereof.

[0037]Examples of growth factors for delivery using the present invention
include, without limitation, growth factors that can be isolated from
native or natural sources, such as from mammalian cells, or can be
prepared synthetically, such as by recombinant DNA techniques or by
various chemical processes. In addition, analogs, fragments, or
derivatives of these factors can be used, provided that they exhibit at
least some of the biological activity of the native molecule. For
example, analogs can be prepared by expression of genes altered by
site-specific mutagenesis or other genetic engineering techniques.

[0038]Examples of anticoagulants for delivery using the present invention
include, without limitation, include warfarin, heparin, Hirudin, and the
like. Examples of factors acting on the immune system include for
delivery using the present invention include, without limitation, factors
which control inflammation and malignant neoplasms and factors which
attack infective microorganisms, such as chemotactic peptides and
bradykinins.

[0039]Examples of viral antigens include, but are not limited to, e.g.,
retroviral antigens such as retroviral antigens from the human
immunodeficiency virus (HIV) antigens such as gene products of the gag,
pol, and env genes, the Nef protein, reverse transcriptase, and other HIV
components; hepatitis viral antigens such as the S, M, and L proteins of
hepatitis B virus, the pre-S antigen of hepatitis B virus, and other
hepatitis, e.g., hepatitis A, B, and C, viral components such as
hepatitis C viral RNA; influenza viral antigens such as hemagglutinin and
neuraminidase and other influenza viral components; measles viral
antigens such as the measles virus fusion protein and other measles virus
components; rubella viral antigens such as proteins E1 and E2 and other
rubella virus components; rotaviral antigens such as VP7sc and other
rotaviral components; cytomegaloviral antigens such as envelope
glycoprotein B and other cytomegaloviral antigen components; respiratory
syncytial viral antigens such as the RSV fusion protein, the M2 protein
and other respiratory syncytial viral antigen components; herpes simplex
viral antigens such as immediate early proteins, glycoprotein D, and
other herpes simplex viral antigen components; varicella zoster viral
antigens such as gpI, gpII, and other varicella zoster viral antigen
components; Japanese encephalitis viral antigens such as proteins E, M-E,
M-E-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitis viral
antigen components; rabies viral antigens such as rabies glycoprotein,
rabies nucleoprotein and other rabies viral antigen components. See
Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M.
(Raven Press, New York, 1991) for additional examples of viral antigens.

[0040]Antigenic targets that may be delivered using the rAb-DC/DC-antigen
vaccines of the present invention include genes encoding antigens such as
viral antigens, bacterial antigens, fungal antigens or parasitic
antigens. Viruses include picornavirus, coronavirus, togavirus,
flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus,
arenavirus, reovirus, retrovirus, papilomavirus, parvovirus, herpesvirus,
poxvirus, hepadnavirus, and spongiform virus. Other viral targets include
influenza, herpes simplex virus 1 and 2, measles, dengue, smallpox, polio
or HIV. Pathogens include trypanosomes, tapeworms, roundworms,
helminthes, malaria. Tumor markers, such as fetal antigen or prostate
specific antigen, may be targeted in this manner. Other examples include:
HIV env proteins and hepatitis B surface antigen. Administration of a
vector according to the present invention for vaccination purposes would
require that the vector-associated antigens be sufficiently
non-immunogenic to enable long term expression of the transgene, for
which a strong immune response would be desired. In some cases,
vaccination of an individual may only be required infrequently, such as
yearly or biennially, and provide long term immunologic protection
against the infectious agent. Specific examples of organisms, allergens
and nucleic and amino sequences for use in vectors and ultimately as
antigens with the present invention may be found in U.S. Pat. No.
6,541,011, relevant portions incorporated herein by reference, in
particular, the tables that match organisms and specific sequences that
may be used with the present invention.

[0042]Fungal antigens for use with compositions and methods of the
invention include, but are not limited to, e.g., candida fungal antigen
components; histoplasma fungal antigens such as heat shock protein 60
(HSP60) and other histoplasma fungal antigen components; cryptococcal
fungal antigens such as capsular polysaccharides and other cryptococcal
fungal antigen components; coccidiodes fungal antigens such as spherule
antigens and other coccidiodes fungal antigen components; and tinea
fungal antigens such as trichophytin and other coccidiodes fungal antigen
components.

[0043]Examples of protozoal and other parasitic antigens include, but are
not limited to, e.g., plasmodium falciparum antigens such as merozoite
surface antigens, sporozoite surface antigens, circumsporozoite antigens,
gametocyte/gamete surface antigens, blood-stage antigen pf 155/RESA and
other plasmodial antigen components; toxoplasma antigens such as SAG-1,
p30 and other toxoplasmal antigen components; schistosomae antigens such
as glutathione-S-transferase, paramyosin, and other schistosomal antigen
components; leishmania major and other leishmaniae antigens such as gp63,
lipophosphoglycan and its associated protein and other leishmanial
antigen components; and trypanosoma cruzi antigens such as the 75-77 kDa
antigen, the 56 kDa antigen and other trypanosomal antigen components.

[0045]Target antigens on cell surfaces for delivery includes those
characteristic of tumor antigens typically will be derived from the cell
surface, cytoplasm, nucleus, organelles and the like of cells of tumor
tissue. Examples of tumor targets for the antibody portion of the present
invention include, without limitation, hematological cancers such as
leukemias and lymphomas, neurological tumors such as astrocytomas or
glioblastomas, melanoma, breast cancer, lung cancer, head and neck
cancer, gastrointestinal tumors such as gastric or colon cancer, liver
cancer, pancreatic cancer, genitourinary tumors such cervix, uterus,
ovarian cancer, vaginal cancer, testicular cancer, prostate cancer or
penile cancer, bone tumors, vascular tumors, or cancers of the lip,
nasopharynx, pharynx and oral cavity, esophagus, rectum, gall bladder,
biliary tree, larynx, lung and bronchus, bladder, kidney, brain and other
parts of the nervous system, thyroid, Hodgkin's disease, non-Hodgkin's
lymphoma, multiple myeloma and leukemia.

[0046]Examples of antigens that may be delivered alone or in combination
to immune cells for antigen presentation using the present invention
include tumor proteins, e.g., mutated oncogenes; viral proteins
associated with tumors; and tumor mucins and glycolipids. The antigens
may be viral proteins associated with tumors would be those from the
classes of viruses noted above. Certain antigens may be characteristic of
tumors (one subset being proteins not usually expressed by a tumor
precursor cell), or may be a protein which is normally expressed in a
tumor precursor cell, but having a mutation characteristic of a tumor.
Other antigens include mutant variant(s) of the normal protein having an
altered activity or subcellular distribution, e.g., mutations of genes
giving rise to tumor antigens.

[0047]Specific non-limiting examples of tumor antigens include: CEA,
prostate specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and
12, MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides,
ras, myc, tyrosinase, MART (melanoma antigen), Pmel 17(gp 100), GnT-V
intron V sequence (N-acetylglucoaminyltransferase V intron V sequence),
Prostate Ca psm, PRAME (melanoma antigen), β-catenin, MUM-1-B
(melanoma ubiquitous mutated gene product), GAGE (melanoma antigen) 1,
BAGE (melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virus
nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53,
lung resistance protein (LRP), Bcl-2, and Ki-67. In addition, the
immunogenic molecule can be an autoantigen involved in the initiation
and/or propagation of an autoimmune disease, the pathology of which is
largely due to the activity of antibodies specific for a molecule
expressed by the relevant target organ, tissue, or cells, e.g., SLE or
MG. In such diseases, it can be desirable to direct an ongoing
antibody-mediated (i.e., a Th2-type) immune response to the relevant
autoantigen towards a cellular (i.e., a Th1-type) immune response.
Alternatively, it can be desirable to prevent onset of or decrease the
level of a Th2 response to the autoantigen in a subject not having, but
who is suspected of being susceptible to, the relevant autoimmune disease
by prophylactically inducing a Th1 response to the appropriate
autoantigen. Autoantigens of interest include, without limitation: (a)
with respect to SLE, the Smith protein, RNP ribonucleoprotein, and the
SS-A and SS--B proteins; and (b) with respect to MG, the acetylcholine
receptor. Examples of other miscellaneous antigens involved in one or
more types of autoimmune response include, e.g., endogenous hormones such
as luteinizing hormone, follicular stimulating hormone, testosterone,
growth hormone, prolactin, and other hormones.

[0049]As used herein, the term "epitope(s)" refer to a peptide or protein
antigen that includes a primary, secondary or tertiary structure similar
to an epitope located within any of a number of pathogen polypeptides
encoded by the pathogen DNA or RNA. The level of similarity will
generally be to such a degree that monoclonal or polyclonal antibodies
directed against such polypeptides will also bind to, react with, or
otherwise recognize, the peptide or protein antigen. Various immunoassay
methods may be employed in conjunction with such antibodies, such as, for
example, Western blotting, ELISA, RIA, and the like, all of which are
known to those of skill in the art. The identification of pathogen
epitopes, and/or their functional equivalents, suitable for use in
vaccines is part of the present invention. Once isolated and identified,
one may readily obtain functional equivalents. For example, one may
employ the methods of Hopp, as taught in U.S. Pat. No. 4,554,101,
incorporated herein by reference, which teaches the identification and
preparation of epitopes from amino acid sequences on the basis of
hydrophilicity. The methods described in several other papers, and
software programs based thereon, can also be used to identify epitopic
core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al.,
1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these
"epitopic core sequences" may then be readily incorporated into peptides,
either through the application of peptide synthesis or recombinant
technology.

[0050]The preparation of vaccine compositions that includes the nucleic
acids that encode antigens of the invention as the active ingredient, may
be prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid prior to
infection can also be prepared. The preparation may be emulsified,
encapsulated in liposomes. The active immunogenic ingredients are often
mixed with carriers which are pharmaceutically acceptable and compatible
with the active ingredient.

[0051]The term "pharmaceutically acceptable carrier" refers to a carrier
that does not cause an allergic reaction or other untoward effect in
subjects to whom it is administered. Suitable pharmaceutically acceptable
carriers include, for example, one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol, or the like and
combinations thereof. In addition, if desired, the vaccine can contain
minor amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, and/or adjuvants which enhance the
effectiveness of the vaccine. Examples of adjuvants that may be effective
include but are not limited to: aluminum hydroxide,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, which
contains three components extracted from bacteria, monophosphoryl lipid
A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion. Other examples of adjuvants include DDA
(dimethyldioctadecylammonium bromide), Freund's complete and incomplete
adjuvants and QuilA. In addition, immune modulating substances such as
lymphokines (e.g., IFN-γ, IL-2 and IL-12) or synthetic IFN-γ
inducers such as poly I:C can be used in combination with adjuvants
described herein.

[0052]Pharmaceutical products that may include a naked polynucleotide with
a single or multiple copies of the specific nucleotide sequences that
bind to specific DNA-binding sites of the apolipoproteins present on
plasma lipoproteins as described in the current invention. The
polynucleotide may encode a biologically active peptide, antisense RNA,
or ribozyme and will be provided in a physiologically acceptable
administrable form. Another pharmaceutical product that may spring from
the current invention may include a highly purified plasma lipoprotein
fraction, isolated according to the methodology, described herein from
either the patients blood or other source, and a polynucleotide
containing single or multiple copies of the specific nucleotide sequences
that bind to specific DNA-binding sites of the apolipoproteins present on
plasma lipoproteins, prebound to the purified lipoprotein fraction in a
physiologically acceptable, administrable form.

[0053]Yet another pharmaceutical product may include a highly purified
plasma lipoprotein fraction which contains recombinant apolipoprotein
fragments containing single or multiple copies of specific DNA-binding
motifs, prebound to a polynucleotide containing single or multiple copies
of the specific nucleotide sequences, in a physiologically acceptable
administrable form. Yet another pharmaceutical product may include a
highly purified plasma lipoprotein fraction which contains recombinant
apolipoprotein fragments containing single or multiple copies of specific
DNA-binding motifs, prebound to a polynucleotide containing single or
multiple copies of the specific nucleotide sequences, in a
physiologically acceptable administrable form.

[0054]The dosage to be administered depends to a great extent on the body
weight and physical condition of the subject being treated as well as the
route of administration and frequency of treatment. A pharmaceutical
composition that includes the naked polynucleotide prebound to a highly
purified lipoprotein fraction may be administered in amounts ranging from
1 μg to 1 mg polynucleotide and 1 μg to 100 mg protein.

[0055]Administration of an rAb and rAb complexes a patient will follow
general protocols for the administration of chemotherapeutics, taking
into account the toxicity, if any, of the vector. It is anticipated that
the treatment cycles would be repeated as necessary. It also is
contemplated that various standard therapies, as well as surgical
intervention, may be applied in combination with the described gene
therapy.

[0056]Where clinical application of a gene therapy is contemplated, it
will be necessary to prepare the complex as a pharmaceutical composition
appropriate for the intended application. Generally this will entail
preparing a pharmaceutical composition that is essentially free of
pyrogens, as well as any other impurities that could be harmful to humans
or animals. One also will generally desire to employ appropriate salts
and buffers to render the complex stable and allow for complex uptake by
target cells.

[0057]Aqueous compositions of the present invention may include an
effective amount of the compound, dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. Such compositions
can also be referred to as inocula. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except insofar
as any conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is contemplated.
Supplementary active ingredients also can be incorporated into the
compositions. The compositions of the present invention may include
classic pharmaceutical preparations. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations contain
a preservative to prevent the growth of microorganisms.

[0058]Disease States. Depending on the particular disease to be treated,
administration of therapeutic compositions according to the present
invention will be via any common route so long as the target tissue is
available via that route in order to maximize the delivery of antigen to
a site for maximum (or in some cases minimum) immune response.
Administration will generally be by orthotopic, intradermal,
subcutaneous, intramuscular, intraperitoneal or intravenous injection.
Other areas for delivery include: oral, nasal, buccal, rectal, vaginal or
topical. Topical administration would be particularly advantageous for
treatment of skin cancers. Such compositions would normally be
administered as pharmaceutically acceptable compositions that include
physiologically acceptable carriers, buffers or other excipients.

[0059]Vaccine or treatment compositions of the invention may be
administered parenterally, by injection, for example, either
subcutaneously or intramuscularly. Additional formulations which are
suitable for other modes of administration include suppositories, and in
some cases, oral formulations or formulations suitable for distribution
as aerosols. In the case of the oral formulations, the manipulation of
T-cell subsets employing adjuvants, antigen packaging, or the addition of
individual cytokines to various formulation that result in improved oral
vaccines with optimized immune responses. For suppositories, traditional
binders and carriers may include, for example, polyalkylene glycols or
triglycerides; such suppositories may be formed from mixtures containing
the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral
formulations include such normally employed excipients as, for example,
pharmaceutical grades of mannitol, lactose, starch magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate, and the like. These
compositions take the form of solutions, suspensions, tablets, pills,
capsules, sustained release formulations or powders and contain 10%-95%
of active ingredient, preferably 25-70%.

[0060]The antigen encoding nucleic acids of the invention may be
formulated into the vaccine or treatment compositions as neutral or salt
forms. Pharmaceutically acceptable salts include the acid addition salts
(formed with free amino groups of the peptide) and which are formed with
inorganic acids such as, for example, hydrochloric or phosphoric acids,
or with organic acids such as acetic, oxalic, tartaric, maleic, and the
like. Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0061]Vaccine or treatment compositions are administered in a manner
compatible with the dosage formulation, and in such amount as will be
prophylactically and/or therapeutically effective. The quantity to be
administered depends on the subject to be treated, including, e.g.,
capacity of the subject's immune system to synthesize antibodies, and the
degree of protection or treatment desired. Suitable dosage ranges are of
the order of several hundred micrograms active ingredient per vaccination
with a range from about 0.1 mg to 1000 mg, such as in the range from
about 1 mg to 300 mg, and preferably in the range from about 10 mg to 50
mg. Suitable regiments for initial administration and booster shots are
also variable but are typified by an initial administration followed by
subsequent inoculations or other administrations. Precise amounts of
active ingredient required to be administered depend on the judgment of
the practitioner and may be peculiar to each subject. It will be apparent
to those of skill in the art that the therapeutically effective amount of
nucleic acid molecule or fusion polypeptides of this invention will
depend, inter alia, upon the administration schedule, the unit dose of
antigen administered, whether the nucleic acid molecule or fusion
polypeptide is administered in combination with other therapeutic agents,
the immune status and health of the recipient, and the therapeutic
activity of the particular nucleic acid molecule or fusion polypeptide.

[0062]The compositions can be given in a single dose schedule or in a
multiple dose schedule. A multiple dose schedule is one in which a
primary course of vaccination may include, e.g., 1-10 separate doses,
followed by other doses given at subsequent time intervals required to
maintain and or reinforce the immune response, for example, at 1-4 months
for a second dose, and if needed, a subsequent dose(s) after several
months. Periodic boosters at intervals of 1-5 years, usually 3 years, are
desirable to maintain the desired levels of protective immunity. The
course of the immunization can be followed by in vitro proliferation
assays of peripheral blood lymphocytes (PBLs) co-cultured with ESAT6 or
ST-CF, and by measuring the levels of IFN-γ released from the
primed lymphocytes. The assays may be performed using conventional
labels, such as radionucleotides, enzymes, fluorescent labels and the
like. These techniques are known to one skilled in the art and can be
found in U.S. Pat. Nos. 3,791,932, 4,174,384 and 3,949,064, relevant
portions incorporated by reference.

[0063]The modular rAb carrier and/or conjugated rAb
carrier-(cohesion/dockerin and/or dockerin-cohesin)-antigen complex
(rAb-DC/DC-antigen vaccine) may be provided in one or more "unit doses"
depending on whether the nucleic acid vectors are used, the final
purified proteins, or the final vaccine form is used. Unit dose is
defined as containing a predetermined-quantity of the therapeutic
composition calculated to produce the desired responses in association
with its administration, i.e., the appropriate route and treatment
regimen. The quantity to be administered, and the particular route and
formulation, are within the skill of those in the clinical arts. The
subject to be treated may also be evaluated, in particular, the state of
the subject's immune system and the protection desired. A unit dose need
not be administered as a single injection but may include continuous
infusion over a set period of time. Unit dose of the present invention
may conveniently may be described in terms of DNA/kg (or protein/Kg) body
weight, with ranges between about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1,
10, 50, 100, 1,000 or more mg/DNA or protein/kg body weight are
administered. Likewise the amount of rAb-DC/DC-antigen vaccine delivered
can vary from about 0.2 to about 8.0 mg/kg body weight. Thus, in
particular embodiments, 0.4 mg, 0.5 mg, 0.8 mg, 1.0 mg, 1.5 mg, 2.0 mg,
2.5 mg, 3.0 mg, 4.0 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg and 7.5 mg
of the vaccine may be delivered to an individual in vivo. The dosage of
rAb-DC/DC-antigen vaccine to be administered depends to a great extent on
the weight and physical condition of the subject being treated as well as
the route of administration and the frequency of treatment. A
pharmaceutical composition that includes a naked polynucleotide prebound
to a liposomal or viral delivery vector may be administered in amounts
ranging from 1 μg to 1 mg polynucleotide to 1 μg to 100 mg protein.
Thus, particular compositions may include between about 1 μg, 5 μg,
10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80
μg, 100 μg, 150 μg, 200 μg, 250 μg, 500 μg, 600 μg,
700 μg, 800 μg, 900 μg or 1,000 μg polynucleotide or protein
that is bound independently to 1 μg, 5 μg, 10 μg, 20 μg, 3.0
μg, 40 μg 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150
μg, 200 μg, 250 μg, 500 μg, 600 μg, 700 μg, 800 μg,
900 μg, 1 mg, 1.5 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg,
70 mg, 80 mg, 90 mg or 100 mg vector.

[0064]The present invention was tested in an in vitro cellular system that
measures immune stimulation of human Flu-specific T cells by dendritic
cells to which Flu antigen has been targeted. The results shown herein
demonstrate the specific expansion of such antigen specific cells at
doses of the antigen which are by themselves ineffective in this system.

[0065]The present invention may also be used to make a modular rAb carrier
that is, e.g., a recombinant humanized mAb (directed to a specific human
dendritic cell receptor) complexed with protective antigens from Ricin,
Anthrax toxin, and Staphylococcus B enterotoxin. The potential market for
this entity is vaccination of all military personnel and stored vaccine
held in reserve to administer to large population centers in response to
any biothreat related to these agents. The invention has broad
application to the design of vaccines in general, both for human and
animal use. Industries of interest include the pharmaceutical and
biotechnology industries.

[0066]The present invention includes compositions and methods, including
vaccines, that specifically target (deliver) antigens to
antigen-presenting cells (APCs) for the purpose of eliciting potent and
broad immune responses directed against the antigen. These compositions
evoke protective or therapeutic immune responses against the agent
(pathogen or cancer) from which the antigen was derived. In addition the
invention creates agents that are directly, or in concert with other
agents, therapeutic through their specific engagement of a receptor
called DC-ASGPR that is expressed on antigen-presenting cells.

[0067]The novel recombinant humanized mAb (directed to the specific human
dendritic cell receptor DC-ASGPR) fused through the antibody (Ab) heavy
chain to antigens known or suspected to encode protective antigens. These
include as examples for vaccination against various
agents--hemagglutinins from Influenza H5N1; HIV gag from attenuated
toxins from Ricin, Anthrax toxin, and Staphylococcus B enterotoxin;
`strings` of antigenic peptides from melanona antigens, etc. The present
invention may be used as a preventative or therapeutic vaccination for at
risk or infected patients. The invention has broad application for
vaccination against many diseases and cancers, both for human and animal
use. Industries that can use the present invention include the
pharmaceutical and biotechnological.

[0068]The present invention can be used to target antigens to APC for
vaccination purposes. It is not known which antigen internalizing
receptor will be best suited for this purpose. The invention describes
particularly advantageous features of DC-ASGPR as for this purpose.
Furthermore, the invention shows that engaging DC-ASGPR can be beneficial
in the sense of activating the immune system with highly predicted
significant therapeutic benefit.

[0069]The present invention includes the development of high affinity
monoclonal antibodies against human DC-ASGPR. Receptor ectodomain.hIgG
(human IgGlFc) and AP (human placental alkaline phosphatase) fusion
proteins were produced for immunization of mice and screening of mAbs,
respectively. An expression construct for hDCIR ectodomain.IgG was
described previously (Bates, Fournier et al. 1999) and used the mouse
SLAM (mSLAM) signal peptide to direct secretion (Bendtsen, Nielsen et al.
2004). An expression vector for hDCIR ectodomain. AP was generated using
PCR to amplify AP resides 133-1581 (gb|BC009647|) while adding a proximal
in-frame Xho I site and a distal TGA stop codon and Not I site. This Xho
I--Not I fragment replaced the IgG coding sequence in the above hDCIR
ectodomain.IgG vector. DC-ASGPR ectodomain constructs in the same Ig and
AP vector series contained inserts encoding (bp 484-1251, gi|53832017).
DC-ASGPR fusion proteins were produced using the FreeStyle® 293
Expression System (Invitrogen) according to the manufacturer's protocol
(1 mg total plasmid DNA with 1.3 ml 293 Fectin reagent/L of
transfection). For rAb production, equal amounts of vector encoding the H
and L chain were co-transfected. Transfected cells are cultured for 3
days, the culture supernatant was harvested and fresh media added with
continued incubation for two days. The pooled supernatants were clarified
by filtration. Receptor ectodomain.hIgG was purified by HiTrap protein A
affinity chromatography with elution by 0.1 M glycine pH 2.7 and then
dialyzed versus PBS. rAbs (recombinant antibodies described later) were
purified similarly, by using HiTrap MabSelect® columns. Mouse mAbs
were generated by conventional cell fusion technology. Briefly,
6-week-old BALB/c mice were immunized intraperitonealy with 20 μg of
receptor ectodomain.hIgGFc fusion protein with Ribi adjuvant, then boosts
with 20 μg antigen 10 days and 15 days later. After 3 months, the mice
were boosted again three days prior to taking the spleens. Alternately,
mice were injected in the footpad with 1-10 μg antigen in Ribi
adjuvant every 3-4 days over a 30-40 day period. 3-4 days after a final
boost, draining lymph nodes were harvested. B cells from spleen or lymph
node cells were fused with SP2/O-Ag 14 cells (Shulman, Wilde et al. 1978)
using conventional techniques. ELISA was used to screen hybridoma
supernatants against the receptor ectodomain fusion protein compared to
the fusion partner alone, or versus the receptor ectodomain fused to AP
(Bates, Fournier et al. 1999). Positive wells were then screened in FACS
using 293F cells transiently transfected with expression plasmids
encoding full-length receptor cDNAs. Selected hybridomas were single cell
cloned and expanded in CELLine flasks (Intergra). Hybridoma supernatants
were mixed with an equal volume of 1.5 M glycine, 3 M NaCl, 1×PBS,
pH 7.8 and tumbled with MabSelect resin. The resin was washed with
binding buffer and eluted with 0.1 M glycine, pH 2.7. Following
neutralization with 2 M Tris, mAbs were dialyzed versus PBS.

[0070]Characterization of purified anti-DC-ASGPR monoclonal antibodies by
direct ELISA. the relative affinities of several anti-DC-ASGPR mAbs by
ELISA were determined (i.e., DC-ASGPR.Ig protein is immobilized on the
microplate surface and the antibodies are tested in a dose titration
series for their ability to bind to DC-ASGPR.Ig (as detected by an
anti-mouse IgG.HRP conjugate reagent. In this example, PAB42 and PAB44
show higher affinity binding than other mAbs. The same mAbs fail to bind
significantly to human Ig bound to the microplate surface. This shows
that the mAbs react to the DC-ASGPR ectodomain part of the DC-ASGPR.Ig
fusion protein (data not shown).

[0071]Characterization of purified anti-DC-ASGPR monoclonal antibodies by
indirect ELISA. Next, the relative affinities of several anti-DC-ASGPR
mAbs were determined by ELISA (i.e., anti-DC-ASGPR mAb is immobilized on
the microplate surface and tested in a dose titration series for their
ability to bind to DC-ASGPR.AP reagent. It was found that the
supernatants from the hybridomas listed as: PAB42, PAB44 and PAB54 show
higher affinity binding than other mAbs (data not shown).

[0072]Characterization of anti-DC-ASGPR mAbs by FACS. The panel of mAbs
was also tested by FACS versus 293F cells transfected with expression
plasmid directing synthesis of cell surface DC-ASGPR. Mean fluorescence
intensity of the signal was subtracted from the analogous signal versus
non-transfected 293F cells. By this criterion, the mAbs are able to bind
to specifically to the surface of cells bearing DC-ASGPR. Some mAbs,
e.g., 37A7 appear particularly advantageous in this regard (data not
shown).

[0073]FIGS. 1A to 1D shows that signaling through DC-ASGPR activates DCs.
DCs are the primary immune cells that determine the results of immune
responses, either induction or tolerance, depending on their activation
(15). The role of LLRs in DC activation is not clear yet. Therefore, we
tested whether triggering the LLR DC-ASGPR can result in the activation
of DCs. Both three and six day in vitro cultured GM/IL-4 DCs express
LOX-1, ASGPR, and CLEC-6 (FIG. 1A). Six day DCs were stimulated with mAb
specific to DC-ASGPR, and data in FIG. 1B show that signals through
DC-ASGPR could activate DCs, resulting in the increased expression of
CD86 and HLA-DR. Triggering DC-ASGPR on DCs also resulted in the
increased production of IL-6, MCP-1, IL-12p40, and IL-8 from DCs (FIG.
1C). Other cytokines and chemokines, TNFa, IP-10, MIP-1a, and IL-10, were
also significantly increased (data not shown) by signaling through
DC-ASGPR, suggesting that DC-ASGPR can deliver cellular signals to
activate DCs. Consistently, DCs stimulated with DC-ASGPR specific mAb
expressed increased levels of multiple genes, including co-stimulatory
molecules as well as chemokine and cytokine-related genes (FIG. 1D). The
possible contribution of LLRs in TLR2 and TLR4-mediated immune cell
activation has been described previously (13, 16). We observed that
signals through DC-ASGPR could synergize with signal through CD40 for a
further activation of DCs (FIG. 1E). This is important because LLRs could
serve as co-stimulatory molecules during in vivo DC activation. Taken
together, data in FIG. 1 prove that signaling through DC-ASGPR can
activate DCs and that DC-ASGPR serves as a co-stimulatory molecule for
the activation of DCs. DC-ASGPR engagement during CD40-CD40L interaction
results in dramatically increased production of IL-12p70.

[0074]DCs stimulated through DC-ASGPR induce potent humoral immune
responses. DCs play an important role in humoral immune responses by
providing signals for both T-dependent and T-independent B cell responses
(19-22) and by transferring antigens to B cells (23, 24). In addition to
DCs, signaling through TLR9 as a third signal is necessary for efficient
B cell responses (25, 26).

[0075]Therefore, we tested the role of DC-ASGPR in DCs-mediated humoral
immune responses in the presence of TLR9 ligand, CpG. Six day GM/IL-4 DCs
were stimulated with anti-DC-ASGPR mAb, and then purified B cells were
co-cultured. As shown in FIG. 2A, DCs activated with anti-DC-ASGPR mAb
resulted in remarkably enhanced B cell proliferation (CFSE dilution) and
plasma cell differentiation (CD38+CD20.sup.-), compared to DCs
stimulated with control mAb. CD38+CD20.sup.- B cells have a typical
morphology of plasma cells, but they do not express CD138. The majority
of proliferating cells did not express CCR2, CCR4, CCR6, or CCR7. The
amounts of total immunoglobulins (Igs) produced were measured by ELISA
(FIG. 2B). Consistent with the data in FIGS. 2A, B cells cultured with
anti DC-ASGPR-stimulated DCs resulted in significantly increased
production of total IgM, IgG, and IgA. In addition to the total Igs, we
also observed that DCs activated by triggering DC-ASGPR are more potent
than DCs stimulated with control mAb for the production of
influenza-virus-specific IgM, IgG, and IgA (FIG. 2c) by B cells,
suggesting that DC-ASGPR-mediated DC activation contributes to both total
and antigen specific humoral immune responses. We tested the role of
DC-ASGPR in ex vivo antigen presenting cells (APCs) in humoral immune
responses. Parts of APCs in PBMCs, including CD19+ and CD14+
cells, express DC-ASGPR (Supplementary FIG. 2). PBMCs from buffy coats
were cultured in the plates coated with anti-DC-ASGPR mAb, and the total
Igs and B cell proliferation were measured. Consistent with the data
generated from DCs (FIG. 2A), APCs stimulated through DC-ASGPR resulted
in enhanced B cell proliferation and plasma cell differentiation in the
absence (upper panels in FIG. 2D) or presence (lower panels in FIG. 2D)
of TLR9 ligand. The total IgM, IgG, and IgA were also significantly
increased when PBMCs were cultured in the plates coated with mAb against
DC-ASGPR (FIG. 2e). As shown in FIG. 1, DCs activated by signaling
through DC-ASGPR have matured phenotypes and produce large amounts of
inflammatory cytokines and chemokines, and both matured DC phenotypes and
soluble factors from DCs could contribute to the enhanced B cells
responses (FIG. 2). However, DC-derived B lymphocyte stimulator protein
(BLyS, BAFF) and a proliferation-inducing ligand (APRIL) are also
important molecules by which DCs can directly regulate human B cell
proliferation and function (27-30). Therefore, we tested whether signals
through DC-ASGPR could alter the expression levels of BLyS and APRIL.
Data in FIG. 2D show that DCs stimulated through DC-ASGPR expressed
increased levels of intracellular APRIL as well as APRIL secreted, but
not BLyS (not shown). Expression levels of BLyS and APRIL receptors on B
cells in the mixed cultures were measured, but there was no significant
change (not shown).

[0076]DC-ASGPR contributes to B cell activation and Ig production.
CD19+ B cells express DC-ASGPR (FIG. 3A). Therefore, we tested the
role of DC-ASGPR in B cell activation. Data in FIG. 3B show that B cells
stimulated through DC-ASGPR produced significantly higher amounts of
chemokines. In addition to IL-8 and MIP-1a, slight increases in IL-6 and
TNFα were also observed when B cells were stimulated with the
anti-DC-ASGPR mAb, compared to control mAb. Genes related to cell
activation were also up-regulated (FIG. 3c). B cells produced IgM, IgG,
and IgA when they were stimulated through DC-ASGPR (FIG. 3D), suggesting
that DC-ASGPR could play an important role in the maintenance of normal
immunoglobulin levels in vivo. However, signaling through DC-ASGPR alone
did not induce significant B cell proliferation.

[0077]Role of DC-ASGPR in T cell responses. DCs stimulated through
DC-ASGPR express enhanced levels of co-stimulatory molecules and produce
increased amounts of cytokines and chemokines (see FIG. 1), suggesting
that DC-ASGPR contributes to cellular immune responses as well as humoral
immune responses. This was tested by a mixed lymphocyte reaction (MLR).
Proliferation of purified allogeneic T cells was significantly enhanced
by DCs stimulated with mAb specific for DC-ASGPR (FIG. 4A). DCs activated
through DC-ASGPR could also prime Mart-1-specific CD8 T cells more
efficiently than DC stimulated with control mAb (upper panels in FIG.
4B). More importantly, signaling through DC-ASGPR permitted DCs to
cross-prime Mart-1 peptides to CD8 T cells (lower panels in FIG. 4B).
This indicates that DC-ASGPR plays an important role in enhancing DC
function, resulting in better priming and cross-priming of antigens to
CD8 T cells. The role of DC-ASGPR expressed on the mixture of APCs in
PBMCs in activation of T cell responses is shown in FIG. 4c where PBMCs
stimulated with mAb to DC-ASGPR resulted in an increased frequency of Flu
M1 tetramer specific CD8 T cells compared to DCs stimulated with control
mAb. This enhanced antigen specific CD8 T cell response was supported by
the data in FIG. 4D, showing that DCs stimulated through DC-ASGPR
significantly increase CD4 T cell proliferation.

[0080]Cells and cultures--Monocytes (1×106/ml) from normal
donors were cultured in Cellgenics (France) media containing GM-CSF (100
ng/ml) and IL-4 (50 ng/ml) (R&D, CA). For day three and day six, DCs, the
same amounts of cytokines were supplemented into the media on day one and
day three, respectively. B cells were purified with a negative isolation
kit (BD). CD4 and CD8 T cells were purified with magnetic beads coated
with anti-CD4 or CD8 (Milteniy, Calif.). PBMCs were isolated from Buffy
coats using Percoll® gradients (GE Healthcare UK Ltd, Buckinghamshire,
UK) by density gradient centrifugation. For DC activation,
1×105 DCs were cultured in the mAb-coated 96-well plate for
16-18 h. mAbs (1-2 μg/well) in carbonate buffer, pH 9.4, were
incubated for at least 3 h at 37° C. Culture supernatants were
harvested and cytokines/chemokines were measured by Luminex (Biorad, CA).
For gene analysis, DCs were cultured in the plates coated with mAbs for 8
h. In some experiments, soluble 50 ng/ml of CD40L (R&D, CA) or 50 nM CpG
(InVivogen, CA) was added into the cultures. In the DCs and B cell
co-cultures, 5×103 DCs resuspended in RPMI 1640 with 10% FCS
and antibiotics (Biosource, CA) were first cultured in the plates coated
with mAbs for at least 6 h, and then 1×105 purified autologous
B cells labeled with CFSE (Molecular Probes, OR) were added. In some
experiments, DCs were pulsed with 5 moi (multiplicity of infection) of
heat-inactivated influenza virus (A/PR/8 H1N1) for 2 h, and then mixed
with B cells. For the DCs and T cell co-cultures, 5×103 DCs
were cultured with 1×105 purified autologous CD8 T cells or
mixed allogeneic T cells. Allogeneic T cells were pulsed with 1
μCi/well 3-[H]-thymidine for the final 18 h of incubation, and
then cpm were measured by a μ-counter (Wallac, Minn.).
5×105 PBMCs/well were cultured in the plates coated with mAbs.
The frequency of Mart-1 and Flu M1 specific CD8 T cells was measured by
staining cells with anti-CD8 and tetramers on day ten and day seven of
the cultures, respectively. 10 μM of Mart-1 peptide (ELAGIGILTV) (SEQ
ID NO.: 2) and 20 nM of recombinant protein containing Mart-1 peptides
(see below) were added to the DC and CD8 T cell cultures. 20 nM purified
recombinant Flu M1 protein (see below) was add to the PBMC cultures.

[0081]Monoclonal antibodies--Mouse mAbs were generated by conventional
technology. Briefly, six-week-old BALB/c mice were immunized i.p. with 20
μg of receptor ectodomain.hIgGFc fusion protein with Ribi adjuvant,
then boosts with 20 μg antigen ten days and fifteen days later. After
three months, the mice were boosted again three days prior to taking the
spleens. Alternately, mice were injected in the footpad with 1-10 μg
antigen in Ribi adjuvant every three to four days over a thirty to forty
day period. Three to four days after a final boost, draining lymph nodes
were harvested. B cells from spleen or lymph node cells were fused with
SP2/O-Ag 14 cells. Hybridoma supernatants were screened to analyze Abs to
the receptor ectodomain fusion protein compared to the fusion partner
alone, or the receptor ectodomain fused to alkaline phosphatase (44).
Positive wells were then screened in FACS using 293F cells transiently
transfected with expression plasmids encoding full-length receptor cDNAs.
Selected hybridomas were single cell cloned and expanded in CELLine
flasks (Integra, CA). Hybridoma supernatants were mixed with an equal
volume of 1.5 M glycine, 3 M NaCl, 1×PBS, pH 7.8 and tumbled with
MabSelect resin. The resin was washed with binding buffer and eluted with
0.1 M glycine, pH 2.7. Following neutralization with 2 M Tris, mAbs were
dialyzed versus PBS.

[0082]ELISA--Sandwich ELISA was performed to measure total IgM, IgG, and
IgA as well as flu-specific immunoglobulins (Igs). Standard human serum
(Bethyl) containing known amounts of Igs and human AB serum were used as
standard for total Igs and flu-specific Igs, respectively. Flu specific
Ab titers, units, in samples were defined as dilution factor of AB serum
that shows an identical optical density. The amounts of BAFF and BLyS
were measured by ELISA kits (Bender MedSystem, CA).

[0083]RNA purification and gene analysis--Total RNA extracted with RNeasy
columns (Qiagen), and analyzed with the 2100 Bioanalyser (Agilent).
Biotin-labeled cRNA targets were prepared using the Illumina totalprep
labeling kit (Ambion) and hybridized to Sentrix Human6 BeadChips (46K
transcripts). These microarrays consist of 50mer oligonucleotide probes
attached to 3 μm beads which are lodged into microwells etched at the
surface of a silicon wafer. After staining with Streptavidin-Cy3, the
array surface is imaged using a sub-micron resolution scanner
manufactured by Illumina (Beadstation 500×). A gene expression
analysis software program, GeneSpring, Version 7.1 (Agilent), was used to
perform data analysis.

[0084]Expression and purification of recombinant Flu M1 and MART-1
proteins--PCR was used to amplify the ORF of Influenza A/Puerto
Rico/8/34/Mount Sinai (H1N1) M1 gene while incorporating an Nhe I site
distal to the initiator codon and a Not I site distal to the stop codon.
The digested fragment was cloned into pET-28b(+) (Novagen), placing the
M1 ORF in-frame with a His6 tag, thus encoding His.Flu M1 protein. A
pET28b (+) derivative encoding an N-terminal 169 residue cohesin domain
from C. thermocellum (unpublished) inserted between the Nco I and Nhe I
sites expressed Coh.His. For expression of
Cohesin-Flex-hMART-1-PeptideA-His, the sequence
GACACCACCGAGGCCCGCCACCCCCACCCCCCCGTGACCACCCCCACCACCACCGA
CCGGAAGGGCACCACCGCCGAGGAGCTGGCCGGCATCGGCATCCTGACCGTGATCC
TGGGCGGCAAGCGGACCAACAACAGCACCCCCACCAAGGGCGAATTCTGCAGATA
TCCATCACACTGGCGGCCG (SEQ ID NO.: 3) (encoding
DTTEARHPHPPVTTPTTDRKGTTAEELAGIGILTVILGGKRTNNSTPTKGEFCRYPSHWR P (SEQ ID
NO.: 4)--the shaded residues are the immunodominant HLA-A2-restricted
peptide and the underlined residues surrounding the peptide are from
MART-1) was inserted between the Nhe I and Xho I sites of the above
vector. The proteins were expressed in E. coli strain BL21 (DE3)
(Novagen) or T7 Express (NEB), grown in LB at 37° C. with
selection for kanamycin resistance (40 μg/ml) and shaking at 200
rounds/min to mid log phase growth when 120 mg/L IPTG was added. After
three hours, the cells were harvested by centrifugation and stored at
-80° C. E. coli cells from each 1 L fermentation were resuspended
in 30 ml ice-cold 50 mM Tris, 1 mM EDTA pH 8.0 (buffer B) with 0.1 ml of
protease inhibitor Cocktail II (Calbiochem, CA). The cells were sonicated
on ice 2×5 min at setting 18 (Fisher Sonic Dismembrator 60) with a
5 min rest period and then spun at 17,000 r.p.m. (Sorvall SA-600) for 20
min at 4° C. For His.Flu M1 purification the 50 ml cell lysate
supernatant fraction was passed through 5 ml Q Sepharose beads and 6.25
ml 160 mM Tris, 40 mM imidazole, 4 M NaCl pH 7.9 was added to the Q
Sepharose flow through. This was loaded at 4 ml/min onto a 5 ml HiTrap
chelating HP column charged with Ni++. The column-bound protein was
washed with 20 mM NaPO4, 300 mM NaCl pH 7.6 (buffer D) followed by
another wash with 100 mM H3COONa pH 4.0. Bound protein was eluted
with 100 mM H3COONa pH 4.0. The peak fractions were pooled and
loaded at 4 ml/min onto a 5 ml HiTrap S column equilibrated with 100 mM
H3COONa pH 5.5, and washed with the equilibration buffer followed by
elution with a gradient from 0-1 M NaCl in 50 mM NaPO4 pH 5.5. Peak
fractions eluting at about 500 mM NaCl were pooled. For Coh.Flu M1.H is
purification, cells from 2 L of culture were lysed as above. After
centrifugation, 2.5 ml of Triton X114 was added to the supernatant with
incubation on ice for 5 min. After further incubation at 25° C.
for 5 min, the supernatant was separated from the Triton X114 following
centrifugation at 25° C. The extraction was repeated and the
supernatant was passed through 5 ml of Q Sepharose beads and 6.25 ml 160
mM Tris, 40 mM imidazole, 4 M NaCl pH 7.9 was added to the Q Sepharose
flow through. The protein was then purified by Ni++ chelating
chromatography as described above and eluted with 0-500 mM imidazole in
buffer D.

[0085]Only particular anti-DC-ASGPR mAbs have DC activation
properties--The invention discloses that DC activation is not a general
property of anti-DC-ASGPR antibodies, rather only certain anti-DC-ASGPR
mAbs have this function. FIG. 5 shows that only certain mAbs activate DCS
through the DC-ASGPR, which must be characterized by screening against
actual DCs.

[0087]Engineered recombinant anti-DC-ASGPR recombinant antibody--antigen
fusion proteins ((rAb.antigen) are efficacious prototype vaccines in
vitro--Expression vectors can be constructed with diverse protein coding
sequence e.g., fused in-frame to the H chain coding sequence. For
example, antigens such as Influenza HA5, Influenza M1, HIV gag, or
immuno-dominant peptides from cancer antigens, or cytokines, can be
expressed subsequently as rAb.antigen or rAb.cytokine fusion proteins,
which in the context of this invention, can have utility derived from
using the anti-DC-ASGPR V-region sequence to bring the antigen or
cytokine (or toxin) directly to the surface of the antigen presenting
cell bearing DC-ASGPR. This permits internalization of e.g.,
antigen--sometimes associated with activation of the receptor and ensuing
initiation of therapeutic or protective action (e.g., via initiation of a
potent immune response, or via killing of the targeted cell. An
exemplative prototype vaccine based on this concept could use a H chain
vector such as [mAnti-ASGPR-5F10H-LV-hIgG4H--C-Flex-FluHA5-1-6×His]
or EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMKWVKQSHGKSLEWIGDINPNYGD
TFYNQKFEGKATLTVDKSSRTAYMQLNSLTSEDSAVYYCGRGDYGYFDVWGAGTTVT
VSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV
DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASDTTEPATPTTPVTJDQICIGYH
ANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLL
GNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPK
SSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVL
WGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFW
TILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINS
SMPFHNIHPLTIGECPKYVKSNRLVLAHHHHHH (SEQ ID NO.: 13). The above sequence
corresponds to the chimeric H chain shown already fused via a flexible
linker sequence (shown italicized) to HA-1 domain of avian Flu HA5 (shown
in bold). This can be co-expressed with the corresponding L chain
chimeric sequence already shown above. Similarly, the sequence
[mAnti-ASGPR--49C11--7H-LV-hIgG4H--C-Dockerin]
DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYILFSGSTN
YNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFCARSNYGSFASWGQGTLVTVSAA
KTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS
RWQEGNVFSCSVMHALHNHYTQKSLSLSLGKASNNSPQNEVLYGDVNDDGKVNSTEL
TLLKRYVLKAVSTLPSSKAEKNADVNRDGRVNSSDVTILSRYLIRVIELLPI (SEQ ID NO.: 14) can
be used to express via co-transfection of the corresponding L chain
sequence already shown above a rAb.Dockerin fusion protein.

[0088]FIG. 6 shows that different antigens can be expressed in the context
of a DC-ASGPR rAb. Such an anti-DC-ASGPR rAb.Doc protein can be simply
mixed with any Cohesin.fusion protein to assemble a stable non-covalent
[rAb.Doc:Coh.fusion] complex that functions just as a rAb.fusion protein.
FIG. 6 shows that such a [rAb.Doc:Coh.fusion] complex can focus antigen
to the surface of cells expressing DC-ASGPR. The figure also shows
anti-DC-ASGPR.Doc:Coh.Flu M1 complexes deliver Flu M1 to the surface of
293F cells transfected with DC-ASGPR cDNA. 1 μg/ml (right panel) of
anti-DC-ASGPR.Doc rAb (shown in green) or control hIgG4.Doc rAb (shown in
blue) were incubated with biotinylated Coh.Flu M1 (2 μg/ml) for 1 hr
at R.T. transfected 293F cells were added and incubation continued for 20
min on ice. Cells were then washed and stained with PE-labeled
streptavidin. Cells were then analyzed for PE fluorescence.

[0089]Anti-DC-ASGPR rAb complexed to Flu M1 via Dockerin:Cohesin
interaction targets the antigen to human DCs and results in the expansion
of Flu M1-specific CD8+ T cells--the potential utility of anti-DC-ASGPR
rAbs as devices to deliver antigen to e.g., DC is shown in the figure
below. FIG. 7 shows the dramatic expansion of Flu M1-specific CD8+ cells
is highly predictive of potency of such an agent as a vaccine directed to
eliciting protective immune responses against Flu M1.

[0090]FIG. 8 demonstrated the cross reactivity of the different antibodies
with monkey ASGPR. For pIRES_ASGPR-mon (monkey) was cloned by inserting
the PCR product into NheI-NotI sites of pIRES vector. The sequence of
final product is base on clone 5S10. Most other clones are either similar
to this with one aa difference or identical to this. However, one clone,
5S1, has an A deletion near the 3' end, which generated a shortened and
different C' terminus and maybe used as a second variant. To clone the
monkey ASGPR, the following oligos were used: DC-ASGPR_MoN:
gaattcgctagcCACCATGACATATGAAAACTTCCAAGACTTGGAGAGTGAGGAGAAAGT CCAAGGGG
(SEQ ID NO.: 15); and DC-ASGPR_Mo:
CGAATTCGCGGCCGCTCAGTGACTCTCCTGGCTGGCCTGGGTCAGACCAGCCTCGC AGACCC (SEQ ID
NO.: 16), which is a reverse complement of
GGGTCTGCGAGGCTGGTCTGACCCAGGCCAGCCAGGAGAGTCACTGAGCGGCCGC GAATTCG (SEQ ID
NO.: 17). Sequence comparisons indicate the likely regions of overlap
and, hence, the cross-reactivity, as is known to those if skill in the
art.

[0092]It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method, kit,
reagent, or composition of the invention, and vice versa. Furthermore,
compositions of the invention can be used to achieve methods of the
invention.

[0093]It will be understood that particular embodiments described herein
are shown by way of illustration and not as limitations of the invention.
The principal features of this invention can be employed in various
embodiments without departing from the scope of the invention. Those
skilled in the art will recognize, or be able to ascertain using no more
than routine experimentation, numerous equivalents to the specific
procedures described herein. Such equivalents are considered to be within
the scope of this invention and are covered by the claims.

[0094]All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled in
the art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same extent as
if each individual publication or patent application was specifically and
individually indicated to be incorporated by reference.

[0095]The use of the word "a" or "an" when used in conjunction with the
term "comprising" in the claims and/or the specification may mean "one,"
but it is also consistent with the meaning of "one or more," "at least
one," and "one or more than one." The use of the term "or" in the claims
is used to mean "and/or" unless explicitly indicated to refer to
alternatives only or the alternatives are mutually exclusive, although
the disclosure supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for the
device, the method being employed to determine the value, or the
variation that exists among the study subjects.

[0096]As used in this specification and claim(s), the words "comprising"
(and any form of comprising, such as "comprise" and "comprises"),
"having" (and any form of having, such as "have" and "has"), "including"
(and any form of including, such as "includes" and "include") or
"containing" (and any form of containing, such as "contains" and
"contain") are inclusive or open-ended and do not exclude additional,
unrecited elements or method steps.

[0097]The term "or combinations thereof" as used herein refers to all
permutations and combinations of the listed items preceding the term. For
example, "A, B, C, or combinations thereof" is intended to include at
least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a
particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
Continuing with this example, expressly included are combinations that
contain repeats of one or more item or term, such as BB, AAA, MB, BBC,
AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will
understand that typically there is no limit on the number of items or
terms in any combination, unless otherwise apparent from the context.

[0098]All of the compositions and/or methods disclosed and claimed herein
can be made and executed without undue experimentation in light of the
present disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be applied to
the compositions and/or methods and in the steps or in the sequence of
steps of the method described herein without departing from the concept,
spirit and scope of the invention. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by the
appended claims.